The Impact of lncRNA on Diabetic Kidney Disease: Systematic Review and In Silico Analyses

Background Long noncoding RNA (lncRNA) is involved in the occurrence and development of diabetic kidney disease (DKD). It is necessary to identify the expression of lncRNA from DKD patients through systematic reviews, and then carry out silico analyses to recognize the dysregulated lncRNA and their associated pathways. Methods The study searched Pubmed, Embase, Cochrane Library, WanFang, VIP, CNKI, and CBM to find lncRNA studies on DKD published before March 1, 2021. Systematic review of the literature on this topic was conducted to determine the expression of lncRNA in DKD and non-DKD controls. For the dysregulated lncRNA in DKD patients, silico analysis was performed, and lncRNA2Target v2.0 and starBase were used to search for potential target genes of lncRNA. The Encyclopedia of Genomics (KEGG) pathway enrichment analysis was performed to better identify dysregulated lncRNAs in DKD and determine the associated signal pathways. Results According to the inclusion and exclusion criteria, 28 publications meeting the eligibility criteria were included in the systematic evaluation. A total of 3,394 patients were enrolled in this study, including 1,238 patients in DKD group, and 1,223 diabetic patients, and 933 healthy adults in control group. Compared with the control, there were eight lncRNA disorders in DKD patients (MALAT1, GAS5, MIAT, CASC2, NEAT1, NR_033515, ARAP1-AS2, and ARAP1-AS1). In addition, five lncRNAs (MALAT1, GAS5, MIAT, CASC2, and NEAT1) participated in disease-related signal pathways, indicating their role in DKD. Discussion. This study showed that there were eight lncRNAs in DKD that were persistently dysregulated, especially five lncRNAs which were closely related to the disease. Although systematic review included 28 studies that analyzed the expression of lncRNA in DKD-related tissues, the potential of these dysregulated lncRNAs as biomarkers or therapeutic targets for DKD remains to be further explored. Trial registration. PROSPERO (CRD42021248634).


Background
Diabetic kidney disease (DKD) refers to chronic kidney disease (CKD) caused by diabetes, which is one of the main microvascular complications of diabetes [1]. DKD has now become the main cause of chronic kidney disease and end-stage kidney disease in the world. It is also one of the main causes of death for diabetic patients. Diabetic kidney disease accounts for 40% of end-stage renal disease in the United States and Europe. According to the 2021 Atlas of the International Diabetes Federation, the global prevalence of diabetes among people aged 20-79 is estimated at 10.5% in 2021, rising to 783.2 million by 2045 [2]. Such a large number of diabetic patients means that there will be more diabetic kidney disease patients, and if diabetic patients have renal insufficiency, it will further aggravate microvascular dysfunction and significantly increase the risk of cardiovascular disease and cognitive decline. erefore, it is urgent to investigate the mechanism of diabetic kidney disease and explore new therapeutic targets to delay the progression of the disease and improve the quality of life of patients.
e clinical manifestations of DKD are microalbuminuria, dominant proteinuria, and decreased glomerular filtration rate, which can eventually develop into end-stage renal disease [3]. e pathological features of DKD include glomerular mesangial expansion and hypertrophy, extracellular matrix (ECM) protein deposition, and podocyte apoptosis. Further glomerular sclerosis and tubular interstitial fibrosis will appear, which will eventually lead to renal failure [4,5]. It is well known that DKD is caused by the interaction of environmental, genetic, and epigenetic factors in many aspects. lncRNAs exist stably in body fluids and can be detected. Recent studies have confirmed that some lncRNAs are important regulatory molecules involved in the occurrence and development of DKD, but their working mechanism in DKD is not very clear yet [6].
Noncoding RNAs (ncRNAs) are a type of RNA molecules that do not encode protein. ey are important biological regulatory factors for the body, which regulates gene expression at the transcription and posttranscription level, and play a key role in both physiological and pathological processes. e development process of DKD is also affected. According to their length and function, ncRNAs can be divided into different subtypes, including long ncRNAs (lncRNAs), that is, ncRNAs with a length of more than 200 nucleotides, which do not encode proteins, but exhibit structural and functional heterogeneity. It is known that lncRNAs can be detected in both nuclei and cytoplasm. lncRNAs in nuclei can act on chromatin to regulate gene expression, and lncRNAs in cytoplasm can act on mRNA to regulate the translation process. lncRNAs exert their biological functions as signal molecules, bait molecules, guide molecules, and scaffold molecules by regulating transcription, translation, mRNA shearing, and posttranscriptional modification. lncRNAs regulate gene expression at the transcription, posttranscriptional, and epigenetic levels and participate in cell proliferation, differentiation, and apoptosis. eir abnormal expression is closely related to the occurrence and development of many diseases [7,8].
It has been discovered that noncoding RNAs are involved in the progression of many diseases. e antisense lncRNA-HOX (HOX transcript antisense RNA, HOTAIR) is upregulated in the rat model of diabetic nephropathy [9], and lncRNA myocardial infarction-related transcripts (myocardial Infarction associated transcript, MIAT) are closely related to the onset of myocardial infarction [10]. lncRNAs may play a regulatory role in almost every gene expression stage, but their roles in the human body are still unknown [11]. erefore, in order to further study which lncRNA may be involved in the pathogenesis of DKD and used as a potential biomarker of the disease, the study conducted a systematic review of the literature on this topic. In addition, bioinformatics analysis was performed to investigate the regulatory role of dysregulated lncRNA in the pathogenesis of diabetes mellitus (DM). e flowchart illustrates the detailed flow of the study design ( Figure 1).

Search Strategies.
is systematic review was carried out according to the current guidelines [12,13], and the research protocol was registered on PROSPERO (https://www.crd. york.ac.uk/PROSPERO) with the identification No. of CRD42021248634. PRISMA(2020) checklist was in Supplemental File 1 (Supplemental File 1). English databases (Pubmed, Embase, and Cochrane Library) and Chinese databases (CBM (China Biomedical Database), CNKI (China National Knowledge Infrastructure), VIP (China Science and Technology Database), and Wanfang Database) were searched, and the studies on lncRNA in DKD published before March 1, 2021, were identified. To seek relevant clinical research, the terms below were searched: ("RNA long noncoding" or "untranslated RNA") AND (diabetic nephropathy or diabetic kidney disease or diabetic renal disease). e literature search strategy is in Supplemental File 2 (Supplemental Files 2). Subject words and free words were combined, and different search strategies were taken for Chinese and English language databases. ere were no restrictions on the status or language of the publication.

Inclusion and Exclusion Criteria.
e original articles that analyzed the expression of lncRNAs in patients with DKD and those without DM or those with DM (controls) were enrolled. Studies without control group were excluded. Exclusion criteria: (1) review, nonclinical studies, and case observations; (2) meta-analysis, case reports, and editorials; (3) repeated studies; (4) research data are incorrect, incomplete, or unavailable, or at least one of the main results has not been reported; and (5) research on fuzzy outcome indicators.

Data Extraction.
Two researchers conducted a comprehensive search of relevant databases and independently reviewed the research based on the inclusion criteria. e researchers deleted duplicate records, then screened the titles and abstracts of the remaining search results for relevance, and determined exclusion or further evaluation. If there were differences in the screening process, the two researchers would discuss together or with the help of a third party. e following data were extracted from each study: first author, publication year, research design, sample size, participant characteristics, lncRNA expression, quantitative method, tissue type analyzed, lncRNA expression, the expression profile of lncRNAs in the case group and control group.

Kyoto Encyclopedia of Genes and Genomes (KEGG)
Pathway Enrichment Analysis of lncRNA Target Genes. KEGG pathway enrichment analysis was performed on lncRNA target genes using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), which revealed lncRNA-mRNA-related pathways. Statistical significance was reported using the Benjamini-Hochberg correction.

Protein-Protein Interaction (PPI) Network Construction and Identification of Hub Genes.
e targets genes of lncRNA were put into STRING (https://string-db.org/cgi/ input.pl) to construct the PPI network. Species were limited to Homo sapiens, confidence scores were limited to >0.9 and free spots were hidden. CytoHubba and CytoNCA are network topology analysis plug-ins in Cytoscape, which are used for topology analysis of PPI networks. Degree refers to the number of connections of nodes in the entire network, which reflects the interaction information between nodes. e value of Degree is used as a reference for the importance of the core goal. e target gene in the PPI network is used as a node, the line between the two nodes represents the relevant interaction, and the strength of interaction is represented by the color of the node. Hub genes were defined as genes that played an important role in the network. Cytoscape V3.8.2 was used to construct and visualize PPI networks.

Search Results and Characteristics of the Included Patients.
According to the search strategy, a total of 128 articles were retrieved from the database; 84 duplicate articles were eliminated by the exclusion criteria, and after 16 preliminary screening of titles and abstracts, 28 articles still needed further review. e remaining articles were evaluated in full text, and 28 articles were included in the final analysis. A total of 3,394 patients were enrolled in this study, including 1,238 patients in DKD group, and 1,223 diabetic patients and 933 healthy adults in control group. e flow chart illustrates the literature screening process and results of this systematic review ( Figure 2). e number of lncRNAs with differential expressions between the different study groups and the control group varied from 1 to 858, and the number of samples in the study group ranged from 12 to 120. Among the 28 studies included in this systematic review, the most analyzed samples were serum, plasma, and kidney (Table 1).

Differentially Expressed lncRNAs in DKD.
Eight types of lncRNA disorders (MALAT1, GAS5, MIAT, CASC2, NEAT1, NR_033515, ARAP1-AS2, and ARAP1-AS1) in patients from two or more studies were selected for further evaluation, of which target gene information was found in five. Among them, lncRNA MIAT and lncRNA GAS5 had different trends of regulation. erefore, this may be explained by differences in the types of tissues analyzed (serum, urine, and kidney) ( Table 2).

KEGG Enrichment Analysis of Related Target Genes.
Next, in order to further explore the functional consequences of five lncRNA dysregulations of interest, the   Computational Intelligence and Neuroscience pathway map from the KEGG repository was used to perform a functional enrichment analysis of their protein-coding target genes. As a result, a total of 115 unique pathways were enriched for lncRNA target genes. In addition, as in the five lncRNAs (MALAT1, GAS5, MIAT, CASC2, and NEAT1), no common pathway had been found. Many of the 115 pathways had been confirmed to be involved in the pathogenesis of DKD, such as PI3K/Akt, TNF, HIF-1, AGE/RAGE, apoptosis, and FoxO (Figure 3(b) and 4).

lncRNA-mRNA Interaction.
After data preprocessing and analysis of the two databases, the expression of 1,921 mRNAs from five lncRNAs coregulated by the databases were obtained ( Figure 5, Table 3).

PPI Network.
To distinguish the links between the 1,921 target genes, the study plotted the PPI using data from the STRING database (https://stringg.embl.de/). e     Figure 4: Important KEGG pathway in DKD that may be regulated by persistently dysregulated lncRNAs. e size of the dots represents the number of genes, and the color represents the range of the pathway q-values. e y-axis represents the KEGG pathway, and the x-axis shows the five lncRNAs involved in each selected pathway. Q-value: p value adjusted for multiple testing using the Benjamini-Hochberg method.
Computational Intelligence and Neuroscience 7 edges were discriminated according to the connection score (Table 4, Figure 6).

Biological Analysis of Hub Genes.
e ClueGO tool was used to perform GO and KEGG enrichment analysis on the above target genes. e highly connected proteins in the network are the hub proteins of regulation. e hub proteins in this study include UBA52, TP53, RPS11, RPS6, RPS13, RPS9, RPS16, GNB2L1, RPS5, and RPS3A. ese involve 12 nodes and 56 edges. In addition, KEGG analysis was performed on potential hub genes and the top 10 enriched pathways were identified. e study investigated the role of core targets in gene function and obtained 996, 120, and 84 GO entries, respectively, and the top 10 entries were selected according to the p value (p < 0.01) (Figure 7).

Discussion
At present, some studies have found that the production of "metabolic memory" in kidney cells has led to diabetic nephropathy. e change of epigenetics is the core event in the progression of DKD [43]. In this case, there is a bidirectional regulation between ncRNA and epigenetic modification. lncRNAs are a type of ncRNAs that seem to be involved in the pathogenesis of DKD [44]. erefore, the study conducted a systematic review to further study which lncRNAs are mainly related to DKD. e results indicated that eight lncRNAs were persistently dysregulated in DKD patients. Four kinds of lncRNA disorders (MALAT1, NEAT1, NR_033515, and ARAP1-AS2) were upregulated, while CASC2 and ARAP1-AS1 were downregulated in diabetes cases compared with the control group. Among them, lncRNA MIAT and lncRNA GAS5 showed different trends of regulation.
Growth arrest-specific 5 (GAS5) is differentially expressed in the serum of DKD patients [17,42]. It has been found that GAS5 is differentially expressed in the plasma of diabetic people and mice. e expression of GAS5 is significantly upregulated in the renal cortex of a mouse model of diabetic nephropathy induced by HFD/STZ [51]. GAS5 is located on chromosome lq25 and has 13 exons, producing a series of long noncoding RNA. Part of RNA secondary structure, the encoded transcript, mimics the glucocorticoid response element (GRE), which means that GAS5 can bind to the DNA binding domain of the glucocorticoid receptor. e combination of the two blocks of the glucocorticoid receptor cannot further regulate the transcription of the target gene. GAS5 is also considered to regulate its transcriptional activity through GRE-like regions bound to related hormone receptors (such as androgen, progesterone, and mineralocorticoid receptors) [52]. GAS5 plays an important role in cell stagnation, proliferation, and apoptosis, autophagy and many other biological processes [53], which is also related to the TGFβ/Smad pathway [54].
Myocardial infarction associated translation (MIAT) is differentially expressed in the serum of DKD patients [30,55]. MIAT is an IncRNA produced by transcription of a gene located on chromosome 22ql2.1, which is involved in the regulation process of diabetic retinopathy (DR). Knockout of MIAT can alleviate the dysfunction of endothelial cells and reduce the formation of new blood vessels, blood vessel leakage, and inflammation during DR [56]. In addition, there may be an interaction between IncRNA MIAT and inflammation and apoptosis. Knockdown of the expression of IncRNA MIAT can reduce podocyte damage induced by high glucose and protect podocytes [57]. However, in the review, it was found that the expressions of GAS5 and MIAT were inconsistent, which may be caused by differences in different samples and human samples. Cancer susceptibility candidate 2 (CASC2) is also continuously downregulated in the serum of DKD patients [20,21]. CASC2 is located at 10q26 of chromosome and spans D10S190. It is involved in cell proliferation, apoptosis inhibition, fibrosis, ECM accumulation, and oxidation stress. In vitro experiments showed that the expression of CASC2 was downregulated in podocytes cultured with high glucose. After CASC2 overexpression, the phosphorylation level of JNK1 and the apoptosis rate of podocytes were significantly reduced, and the JNK1 activator could significantly antagonize the apoptosis of podocytes caused by CASC2 overexpression. e inhibitory effect of DN increased the rate of podocyte apoptosis. It can be seen that overexpression of CASC2 inhibits podocyte apoptosis by blocking the JNK pathway, thereby delaying the progression of DN [21]. Long noncoding RNA CASC2 inhibits human mesangial cell proliferation, inflammation, and fibrosis.   Nuclear-enriched abundant transcript 1 (NEAT1) is continuously downregulated in the serum and urine of patients with DKD [31,55]. NEAT1 is a lncRNA located in the nucleus, which is involved in the transcription of many genes, and participates in cell proliferation, inhibition of apoptosis, fibrosis and ECM accumulation, and oxidative stress [58]. It has been found that NEAT1 expression is upregulated in rat kidney tissues and mouse GMCs cultured with high glucose, NEAT1 can promote ECM accumulation and EMT processes and accelerate the process of renal fibrosis [59]. Computer analysis showed that NEAT1 was involved in many pathways related to DM and its complications, including cell cycle and HIF-1. lncRNA NEAT1 accelerates the development and progression of diabetic nephropathy. e expression of NR_033515 in the kidney, peripheral blood, urine, and serum is increased compared with the control group [19,28], which is related to renal fibrosis, and enhance expression levels of fibrogenesis-related gene proteins (P38, ASK1, and ASK1), fibronectin and α-SMA [19]. lncRNA-NR_033515 promotes proliferation, fibrosis, and epithelial-mesenchymal transition in DKD.
In summary, the systematic review showed that eight lncRNAs were continuously dysregulated in DM and DKD patients. e study also clarified the pathways regulated by these lncRNAs and involved in the pathogenesis of DM, such as PI3K/Akt, TNF, HIF-1, AGE/RAGE, insulin resistance, apoptosis, and FoxO. Although this systematic review included 28 studies that analyzed the expression of lncRNA in DKD-related tissues, the involvement of lncRNAs in the pathogenesis of this complex disease remains to be further investigated. Although lncRNAs seem to be a good candidate for DKD biomarkers and therapeutic targets, more studies on the different tissues and cell distribution of these regulatory molecules may further clarify their role in DKD.

Additional Points
Although this systematic review showed that lncRNA always regulated pathways related to DKD, it still has some limitations. First of all, the lncRNAs in some studies did not adopt formal names; therefore, the study may have lost some of the details in the study. Second, different techniques were used to quantify the expression of lncRNA in different studies, and only the expression pattern of lncRNA was provided. erefore, it is impossible to perform a reliable quantitative meta-analysis based on the expression level of lncRNA. Finally, compared with the control group, the eight types of lncRNAs were always dysregulated in DKD patients, but it is not possible to perform hierarchical analysis by tissue type, because the number of studies using different tissues to evaluate the same lncRNA is very small. However, most of the studies included in this systematic review did not report the stages of kidney disease in patients with these DKDs. erefore, here, it is impossible to assess whether different kidney function stages affect results. e findings remain to be further verified.

Data Availability
All data related to the research are included in the article/ Supplementary Material.

Conflicts of Interest
e authors declare that the research was conducted without any potential conflicts of interest.